Enterobacterial phage λ is a temperate phage that infects Escherichia coli and has a lytic-lysogenic life cycle. CI, a λ repressor, regulates the expression of lytic transcripts and acts as a major genetic switch that determines the lysogenic state. To manipulate the genome of phage λ, the CRISPR-Cas9 genome editing system was constructed in lysogenic E. coli MG1655 cells. For instance, we successfully changed cI857 to cIWT in the phage genome through Cas9-mediated single-nucleotide editing. A lytic phage was prepared by introducing an amber mutation in the middle of the cI gene, but it could not lyse lysogenic MG1655 cells. We prepared a phage expressing cI antisense mRNA by reverse substitution of the cI gene. Lysis of λ cI857 lysogenic cells occurred by the infection of the λ cIantisense. These results suggest an effective lysogenic cell control method by a synthetic phage expressing antisense mRNA of the genetic switch gene. It is expected to be applied as a tool to control harmful lysogenic microorganisms.
Keywords: CRISPR-Cas9; antisense; lysogeny; repressor; superinfection immunity.